1. Field of the Invention
The present invention relates to a non-reducing saccharide and its production and use, in particular, to a non-reducing saccharide and less reducing saccharide containing the same which are obtainable by cultivating a microorganism capable of producing non-reducing saccharide-forming enzyme in a nutrient culture medium which contains one or more reducing partial starch hydrolysates with glucose polymerization degrees of 3 or higher, as well as to a process to produce the same and uses thereof.
2. Description of the Prior Art
Trehalose (alpha,alpha-trehalose) has been known from ancient times as a non-reducing saccharide composed of glucose, and as described in Advances in Carbohydrate Chemistry, published by Academic Press Inc., New York, N.Y., USA, Vol.18, pp.201-225 (1963) and Applied and Environmental Microbiology, Vol.56, pp.3,213-3,215 (1990), its trace but extensive distribution is found in microorganisms, mushrooms and insects. Since as is the case with trehalose, non-reducing saccharides cause no amino-carbonyl reactions with substances bearing amino groups such as amino acids and proteins and therefore neither deteriorate nor alter them, the non-reducing saccharides have been deemed to be useful in utilizing and processing such substances with no fears of their browning and deterioration: Thus establishment of processes which would enable their industrial-scale production has long been desired.
There have been known several processes to produce trehalose, for example, those using microorganism cells as disclosed in Japanese Patent Kokai No.154,485/75 and those converting maltose by combination of maltose phosphorylase and trehalose phosphorylase. The former process using microorganism cells is however inadequate for industrial-scale process because the trehalose content in microorganism cells as starting material is generally low, i.e. less than 15 w/w % (the percentages appeared hereinafter mean "w/w %" unless specified otherwise), and the extraction and purification steps for trehalose are very complicated. While the latter process using maltose phosphorylase and trehalose phosphorylase has not been realized in industrial scale due to the demerits that both enzymes commonly act via glucose-1-phosphate and this hinders elevated concentrations for substrates, the yield for trehalose is low because both enzymes irreversibly act in the same reaction system, and further that such reaction system is very difficult to stably maintain and smoothly proceed.
In connection with this, Gekkan Food Chemical (Monthly Food Chemical), "Recent Aspects and Issues in Utilization and Development of Starch", August, pp.67-72 (1992) comments in the corner of "Oligosaccharides" that although trehalose would have very extensive uses, its enzymatic production using any direct saccharide-transferring or hydrolyzing reactions has been deemed to be scientifically impossible at the present time, confirming that the production of trehalose from starch as material using enzymatic reactions has been deemed to be scientifically impossible.
It is known that partial starch hydrolysates, for example, liquefied starch, dextrins and maltooligosaccharides which are all produced from starch, generally exhibit reducing powers due to the reducing end groups in their molecules. Such a partial starch hydrolysate will be designated as a "reducing partial starch hydrolysate" in this specification. The reducing powers of reducing partial starch hydrolysates on dry solid basis are usually expressed by "Dextrose Equivalent" or "DE". Also is known that reducing partial starch hydrolysates with higher DE values, which are generally small molecules, exhibit low viscosities and strong sweetening powers, as well as high reactivities to substances with amino groups such as amino acids and proteins, which cause the amino-carbonyl reaction leading to browning, unpleasant smell and deterioration.
The characteristics of reducing partial starch hydrolysates vary depending on the magnitudes of their DE and therefore the relationship between particular reducing partial starch hydrolysates and their DE values is very important. It has been, however, believed in the art to be impossible to cut off this relationship.
The sole method to cut off the relationship is to change reducing partial starch hydrolysates into non-reducing saccharides, for example, by converting their reducing groups into alcohol groups by high-pressure hydrogenation. This method however needs high-pressure autoclaves, safety facilities and careful control to prevent disasters, as well as consuming large amounts of hydrogen and energy. Further the obtained saccharide alcohols differ from reducing partial starch hydrolysates in the point that reducing partial starch hydrolysates consist of glucose moieties, while the saccharide alcohols consist of glucose and sorbitol and this may cause transient indigestion and diarrhea. Thus there has been in a great demand to establish any methods by which the reducing powers of reducing partial starch hydrolysates are decreased or even eliminated without changing the glucose moieties which compose reducing starch hydrolysates.
To solve these, the present inventors disclose in Japanese Patent Application No.349,216/93 a novel non-reducing saccharide-forming enzyme (referred to as "non-reducing saccharide-forming enzyme" hereinafter) which is capable of forming non-reducing saccharides bearing at their ends trehalose structures from one or more reducing partial starch hydrolysates with glucose polymerization degrees of 3 or higher, thus establishing non-reducing saccharides bearing at their molecular ends trehalose structures and less reducing saccharides containing the same, as well as establishing a process to produce trehalose from these saccharides using the non-reducing saccharide-forming enzyme.
The present inventors also disclose in Japanese Patent Application No.79,291/94 a novel trehalose-releasing enzyme (referred to as "trehalose-releasing enzyme" hereinafter) which specifically hydrolyzes the linkages between the trehalose moieties and other moieties in non-reducing saccharides with glucose polymerization degrees of 3 or higher, as well as establishing a process to produce trehalose at a relatively high yield where the non-reducing saccharide-forming enzyme and trehalose-releasing enzyme are used in combination. It was however found later that, unfavorably, such a process needed an extended time for cultivation of microorganisms to produce enzymes, recovery of the enzymes and enzymatic reaction to convert reducing partial starch hydrolysates into non-reducing saccharides. Thus there is a great demand to establish any processes which would be more readily feasible in the production of non-reducing saccharides from reducing partial starch hydrolysates with less complicated steps including those for preparation of enzymes.